1. Technical Field
The present invention is directed to reagents and methods for performing an immunoassay, particularly a fluorescence polarization immunoassay (FPIA), to determine the presence and/or amount of cotinine in samples, particularly aqueous, fluid biological samples such as urine, blood serum or blood plasma, to a method of making the reagents, and to an immunoassay based on the reagents. More particularly the invention is directed to new haptens, immunogens prepared from the haptens, antibodies raised against the haptens and immunoassays which utilize reagents and methods of the invention.
2. Background
It is believed that a dose-response relationship exists between the number of cigarettes smoked and the risk of developing diseases related to smoking. Insurance companies, surgeons and forensic scientists have shown interest in ways of distinguishing between smokers and non-smokers. Some previous ways have involved the measurement of nicotine and cotinine (a metabolite of nicotine) in biological fluids. Generally, the measurement of urinary cotinine is preferred because (1) in the human body, cotinine is naturally derived only from the metabolism of nicotine, (2) urine is more convenient to collect than blood, (3) cotinine has a half-life of between about 7 and 40 hours, whereas the half-life of nicotine is less than about 30 minutes, and (4) the excretion of cotinine is not as dependent on urinary pH as is nicotine.
The present invention is related to immunoassays, particularly competitive immunoassays, involving reagents and techniques particularly suitable for determining the presence and/or amount of cotinine in biological fluids. The present invention can provide, among others, an advantage of allowing for the determination of the amount of cotinine with minimization of interference from other metabolites of nicotine. The present invention is in part based on new, substantially optically pure haptens, which can be utilized in the preparation of immunogens and/or tracers suitable for use in immunoassays.
3. Background Art
Langone et. al. in Biochemistry, 12(24), pages 5025-5030 (1973), describe the use of racemic 3'-hydroxymethylnicotine and 4'-carboxycotinine as haptens in a radioimmunoassay (RIA) for nicotine and cotinine respectively. However, these provide major drawbacks for immunoassays because of the lack of optical activity of these haptens, and possibly because of the positioning of the linking arm in the case of the cotinine hapten. Racemic haptens ultimately lead to antisera wherein no discrimination between optical antipodes is possible and often results in higher levels of interference.
Japanese Patent Applications, Publication Numbers 61-126083 and 61-126084 are directed to the preparation of 4-aminocotinine and 4-aminonicotine and mention possible utility of these compounds as haptens for immunoassays. While these publications suggest that optical purity is maintained during their preparation according to procedures described in these publications, the synthetic sequences are cumbersome. Additionally, because these derivatives are 4-aminopyridines, their efficacy as true haptens for immunoassays is believed to be severely limited because of their poor nucleophilicity at the amino group and the attendant difficulty in attaching "linking arms" for eventual protein conjugation.
Res. Commun. Chem. Pathol. Pharmacol.,51(3), 393-404 describes the use of 6-aminonicotine. However, as in the work described in Biochemistry, 12(24), pages 5025-5030 (1973), supra, the material described is racemic.
Japanese Patent Number 53/31213 is directed to the use of 4-, 5-, or 6-para-aminobenzamidonicotine derivatives as haptens which are bound to protein through a diazonium linkage. However, the parent aminonicotine used to prepare such materials is racemic. It is also important to point out that antisera raised against compounds having such rigid and heteroatom-containing linking arms have a higher probability of "bridge antibody" production thus lowering the specificity of the derived immunoassay in general.